U.S. patent application number 10/455080 was filed with the patent office on 2004-01-01 for long term expression of lentiviral vectors.
Invention is credited to Chang, Lung-Ji.
Application Number | 20040002158 10/455080 |
Document ID | / |
Family ID | 29783227 |
Filed Date | 2004-01-01 |
United States Patent
Application |
20040002158 |
Kind Code |
A1 |
Chang, Lung-Ji |
January 1, 2004 |
Long term expression of lentiviral vectors
Abstract
Lentiviral vectors that include a transgene and one or more
copies of the cHS4 insulator in a forward or reverse orientation,
when expressed in cells, exhibit prolonged transgene expression
compared to vectors lacking the insulator.
Inventors: |
Chang, Lung-Ji;
(Gainesville, FL) |
Correspondence
Address: |
Stanley A. Kim, Ph.D., Esq.
Akerman Senterfitt
Suite 400
222 Lakeview Avenue
West Palm Beach
FL
33402-3188
US
|
Family ID: |
29783227 |
Appl. No.: |
10/455080 |
Filed: |
June 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60385986 |
Jun 5, 2002 |
|
|
|
60385864 |
Jun 5, 2002 |
|
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Current U.S.
Class: |
435/456 ;
435/235.1; 435/320.1; 435/325; 536/23.72 |
Current CPC
Class: |
C12N 2740/16043
20130101; C12N 15/86 20130101 |
Class at
Publication: |
435/456 ;
435/320.1; 435/235.1; 435/325; 536/23.72 |
International
Class: |
C12N 015/867; C12N
007/00; C07H 021/02; C12N 005/06 |
Goverment Interests
[0002] This invention was made with U.S. government support under
grant numbers 2906420-12 and P50 HL59412 both awarded by the
National Institutes of Health. The U.S. government may have certain
rights in the invention.
Claims
What is claimed is:
1. A nucleic acid molecule comprising a first nucleotide sequence
derived from a lentivirus, a second nucleotide sequence not derived
from the lentivirus, and a third nucleotide sequence that comprises
an insulator.
2. The nucleic acid molecule of claim 1, wherein the insulator is a
cHS4 insulator.
3. The nucleic acid molecule of claim 2, wherein the cHS4 insulator
has the amino acid sequence of SEQ ID NO:1.
4. The nucleic acid molecule of claim 1, wherein the insulator is
in a forward orientation.
5. The nucleic acid molecule of claim 1, wherein the insulator is
in a reverse orientation.
6. The nucleic acid molecule of claim 1, further comprising a
fourth nucleotide sequence that comprises an additional
insulator.
7. The nucleic acid molecule of claim 1, wherein the third
nucleotide sequence that comprises an insulator located upstream
(5' to) of the second nucleotide sequence.
8. The nucleic acid molecule of claim 1, wherein the nucleic acid
molecule is comprised within a plasmid.
9. A cell into which has been introduced a nucleic acid molecule
comprising a first nucleotide sequence derived from a lentivirus, a
second nucleotide sequence not derived from the lentivirus, and a
third nucleotide sequence that comprises an insulator.
10. The cell of claim 9, wherein the insulator is a cHS4
insulator.
11. The cell of claim 10, wherein the cHS4 insulator has the amino
acid sequence of SEQ ID NO:1.
12. The cell of claim 9, wherein the cell is a stem cell.
13. The cell of claim 12, wherein the stem cell is an embryonic
stem cell.
14. A method for promoting long term expression of a lentiviral
vector in a cell, the method comprising the step of introducing
into the cell a nucleic acid molecule comprising a first nucleotide
sequence derived from a lentivirus, a second nucleotide sequence
not derived from the lentivirus, and a third nucleotide sequence
that comprises an insulator.
15. The method of claim 14, wherein the insulator is a cHS4
insulator.
16. The method of claim 15, wherein the cHS4 insulator has the
amino acid sequence of SEQ ID NO: 1.
17. The method of claim 14, wherein the cell is a stem cell.
18. The method of claim 17, wherein the stem cell is an embryonic
stem cell.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of U.S.
provisional patent application No. 60/385,986 filed Jun. 5,
2002.
FIELD OF THE INVENTION
[0003] The invention relates to the fields of molecular biology,
gene therapy, microbiology and virology. More particularly, the
invention relates to compositions and methods for enhancing the
long term expression of lentiviral vectors in cells.
BACKGROUND
[0004] Long term transgene expression is required in gene therapy
applications involving correction of genetic disorders and
permanent phenotype introduction. Retroviral vectors have been the
preferred tool for long term gene transfer because retroviruses
employ a unique proviral chromosomal integration mechanism.
However, ectopic retroviral gene transfer into host cell does not
always ensure long lasting transgene expression. A heterologous
promoter inserted in the murine leukemia oncoretroviral vector
(MLV) may be silenced at different time following infection
depending on the cell type. In embryonic carcinoma (EC), embryonic
stem cells (ES) and early embryos, it has been shown that the
suppression of MLV transgene expression occurs soon after infection
through mechanisms involving loss of unintegrated proviral DNA and
transcriptional silencing. The retroviral transgene silencing is
controlled through negative cellular factors binding to the
repressor binding sites in the long terminal repeats (LTRs) and the
primer binding site (PBS) of the provirus involving DNA methylation
and methylation-independent mechanisms.
[0005] Unlike the silencing observed using oncoretroviral vectors,
several previous reports indicated that silencing was not observed
using lentiviral vectors. In contrast, in the experiments described
herein, silencing was observed using lentiviral vectors. The
identification of this effect using lentiviral vectors indicates
that there is a need to develop silencing-resistant lentiviral
vectors for applications in which long-term transgene expression is
desired, e.g., gene therapy applications.
SUMMARY
[0006] The invention relates to the development of a new method for
promoting the long-term expression of a transgene introduced into a
cell using a lentiviral vector. This method utilizes regulatory
elements known as "insulators" to prevent the silencing effect by
shielding the transgene promoter from the influence of neighboring
regulatory elements.
[0007] The invention also relates to lentiviral constructs that
incorporate an insulator as well as a transgene. A particularly
preferred insulator for use in the invention is the chicken
.beta.-globin HS4 insulator known as cHS4 (or simply HS4). This
insulator was incorporated into lentiviral vectors and evaluated
using two different cell types: TE671 (human rhabdomyosarcoma) and
P 19 (embryonic stem cell). Increasing copy number of HS4 cloned in
the viral LTR appeared to moderately interfere with the virus
production. Without the insulator, a silencing effect in lentiviral
transgene expression was observed in transduced TE671 cells after
15 passages, and the same effect was observed in P19 cells only
after 2 passages. Lentiviral vectors with the HS4 insertion in
either orientation, however, displayed significantly protection of
transgene expression in both types of cells.
[0008] Accordingly, the invention features a nucleic acid molecule
that include a first nucleotide sequence derived from a lentivirus,
a second nucleotide sequence not derived from the lentivirus, and
third nucleotide sequence that includes an insulator. The nucleic
acid of the invention can be included in a plasmid. In other
variations, an insulator is located upstream (5' to) of the second
nucleotide sequence.
[0009] Also within the invention is a cell into which a nucleic
acid molecule of the invention has been introduced. The cell can be
a stem cell such as an embryonic stem cell.
[0010] Another aspect of the invention is a method for promoting
long term expression of a lentiviral vector in a cell. This method
includes the step of introducing a nucleic acid molecule of the
invention into the cell.
[0011] In the nucleic acid molecule, cell, and method of the
invention, the insulator(s) can be a cHS4 insulator such as the
cHS4 insulator that has the amino acid sequence of SEQ ID NO:1. The
insulator(s) can be in a forward or reverse orientation.
[0012] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Commonly
understood definitions of molecular biology terms can be found in
Rieger et al., Glossary of Genetics: Classical and Molecular, 5th
edition, Springer-Verlag: New York, 1991; and Lewin, Genes V,
Oxford University Press: New York, 1994. Commonly understood
definitions of virology terms can be found in Granoff and Webster,
Encyclopedia of Virology, 2nd edition, Academic Press: San Diego,
Calif., 1999; and Tidona and Darai, The Springer Index of Viruses,
1st edition, Springer-Verlag: New York, 2002. Commonly understood
definitions of microbiology can be found in Singleton and
Sainsbury, Dictionary of Microbiology and Molecular Biology, 3rd
edition, John Wiley & Sons: New York, 2002.
[0013] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, suitable methods and materials are described
below. All publications, patent applications, patents and other
references mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions will control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A is a series of highly schematic maps of plasmid
constructs used for generating HIV-1 derived lentiviral
vectors.
[0015] FIG. 1B is a nucleotide sequence of the cHS4 insulator used
in the studies described below. The sequence of a variant of the
cHS4 that was used in other studies is shown by the editorial
markings below the main sequence. These include several
substitutions, on deletion, and two insertions.
[0016] FIG. 1C is highly schematic overview of a making a proviral
DNA from plasmid constructs according to the invention.
[0017] FIG. 2 is two graphs showing the results of a study on the
expression of a nLacZ reporter gene on TE671 cells. A=2 MOI; B=5
MOI.
[0018] FIG. 3 is a graph showing the difference in decrease of
expression of a transgene for different lentiviral constructs.
[0019] FIG. 4 is two graphs showing the results of a study on the
expression of a nLacZ reporter gene on P19 cells. A=2 MOI; B=5
MOI.
[0020] FIG. 5 is a graph showing the effect of the use of an
insulator on P19 cells.
DETAILED DESCRIPTION
[0021] The invention provides methods and compositions for
promoting the long-term expression of a transgene introduced into a
cell using a lentiviral vector. In the embodiments described below,
a number of lentiviral vectors based on human immunodeficiency
virus type 1 (HIV-1) were constructed. These included one or more
copies of a cHS4 insulator in a forward or reverse orientation. The
long term expression of these HS4 lentiviral vectors was studied in
two different cell types: TE671 (human rhabdomyosarcoma) and P19
(embryonic carcinoma cells). Increasing copy number of HS4 in the
LTR appeared to moderately interfere with the virus production.
Without the insulator, a silencing effect in lentiviral transgene
expression was observed in transduced TE671 cells after 15
passages, and in P19 cells only after 2 passages. Lentiviral
vectors with the HS4 insertion in either orientation, however,
displayed significant protection of transgene expression in both
cell types.
[0022] The below described preferred embodiments illustrate
adaptations of these compositions and methods. Nonetheless, from
the description of these embodiments, other aspects of the
invention can be made and/or practiced based on the description
provided below.
Biological Methods
[0023] Methods involving conventional molecular biology techniques
are described herein. Such techniques are generally known in the
art and are described in detail in methodology treatises such as
Molecular Cloning, 3rd Edition, Sambrook and Russell, Cold Spring
Harbor Press, 2001; and Current Protocols in Molecular Biology, ed.
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
1992 (with periodic updates). Various techniques using polymerase
chain reaction (PCR) are described, e.g., in Innis et al., PCR
Protocols: A Guide to Methods and Applications, Academic Press: San
Diego, 1990. PCR-primer pairs can be derived from known sequences
by known techniques such as using computer programs intended for
that purpose (e.g., Primer, Version 0.5, .COPYRGT.1991, Whitehead
Institute for Biomedical Research, Cambridge, Mass.). Methods for
chemical synthesis of nucleic acids are discussed, for example, in
Beaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and
Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemical
synthesis of nucleic acids can be performed, for example, on
commercial automated oligonucleotide synthesizers. Immunological
methods (e.g., preparation of antigen-specific antibodies,
immunoprecipitation, and immunoblotting) are described, e.g., in
Current Protocols in Immunology, ed. Coligan et al., John Wiley
& Sons, New York, 1991; and Methods of Immunological Analysis,
ed. Masseyeff et al., John Wiley & Sons, New York, 1992.
Conventional methods of gene transfer and gene therapy can also be
adapted for use in the present invention. See, e.g., Gene Therapy:
Principles and Applications, ed. T. Blackenstein, Springer Verlag,
1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed.
P. D. Robbins, Humana Press, 1997; and Retro-vectors for Human Gene
Therapy, ed. C. P. Hodgson, Springer Verlag, 1996.
Lentiviral Vectors
[0024] A number of different lentiviral vectors are known including
those based on naturally occurring lentiviruses such as HIV-1,
HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency
virus (FIV), bovine immunodeficiency virus (BIV) and others. See
U.S. Pat. No. 6,207,455. Although the invention is described using
HIV-1 based vectors, other vectors derived from other lentiviruses
might also be used by adapting the information described herein.
Because of the many advantages HIV-1 based vectors provide for gene
therapy applications, these are presently preferred. See U.S. Pat.
No. 6,531,123.
[0025] To render HIV-1 derived vectors safe and efficient for gene
therapy applications, it is desirable to (1) delete a maximum
amount of the virus sequence to avoid the production of wild type
virus by recombination without interfering with the virus efficacy
and (2) insert heterologous sequences to increase the efficacy of
the vector. For example, because efficient synthesis of HIV-1
Gag-Pol requires tat activation of the LTR and the interaction of
Rev-RRE to mediate nuclear export of mRNA, these functions should
be retained. On the other hand, because the accessory gene
functions of vif, vpr, vpu and nef have been shown to be
dispensable for viral replication, one or more of these might be
deleted.
[0026] The lentiviral vectors of the invention might also be
pseudotyped, e.g., to overcome restricted host cell tropism. For
example, lentiviral vectors pseudotyped with vesicular stomatitis
virus G (VSV-G) viral envelopes might be used. In addition, the
potential risk of wild type recombination can be reduced by
designing a three-plasmid co-transfection strategy for vector
production. For example, referring to FIG. 1A, a three-plasmid
design includes a helper construct, pNHP, that encodes the gag-pol
(necessary viral proteins), a transducing vector construct, pTV,
that encodes the viral genome which carries a foreign gene cassette
(reporter gene), and a VSV-G envelope expression plasmid,
pHEF-VSV-G. To increase vector titer in the system, an additional
eukaryotic expression plasmid (e.g., a transactivator plasmid
construct such as pCEP4-tat) might also be utilized.
[0027] To enhance safety, a self-inactivating (SIN) lentiviral
vector might also be used. For example, a SIN lentiviral vector can
be made by inactivating the 3' U3 promoter and deleting of all the
3' U3 sequence except the 5' integration attachment site which is
important for the integration into host chromosome. A particularly
preferred construct for designing vectors of the invention is pTY
shown in FIG. 1A.
Insulators
[0028] Methods and compositions of the invention utilize insulators
to promote long-term expression of a transgene in a cell by
preventing the silencing effect caused by other regulatory
elements. The insulator used in the embodiments described herein is
a chicken HS4 insulator element (cHS4). The amino acid sequence of
the particular cHS4 is provided herein as SEQ ID NO:1; although
other versions of cHS4 that can serve as an insulator are known
(see, e.g., Chung et al., Proc. Natl. Acad. Sci. USA, 94:575-580,
1997). In addition to cHS4, a number of other insulators are known.
For a review, see Pannell et al., Rev. Med. Virol. 11:205-217,
2001. These might also be used in designing vectors for use in the
invention. For example, the scs (scs sequence flanking the 87A1
hsp70 locus), BEAD-1, and gypsy (340 bp fragment from the gypsy
retrotransposon) insulators might be used. See Modin et al., J.
Virol. 74:11697-11707, 2000; Pamell et al., EMBO J. 19:5864-5874,
2000; and Biochem. Biophys. Res. Commun. 284:987-992, 2001.
EXAMPLES
Example 1
Materials and Methods
[0029] As shown in FIG. 1C, three different clones were generated:
(1) pTYcHS4-EFnLacZ forward, a construct that includes one copy of
the cHS4 fragment of SEQ ID NO:1 in forward orientation; (2)
pTYcHS4-EFnLacZ reverse, a construct that includes one copy of the
cHS4 fragment of SEQ ID NO:1 in reverse orientation; and (3)
pTYcHS4-EFnLacZ2xReverse, a construct that includes two copies of
the cHS4 fragment of SEQ ID NO:1 in reverse orientation. After
confirmation by DNA sequencing, these plasmids were retransformed
and produced in a large scale and pure quality.
[0030] Cell Culture. TE671 cells were cultured in Dubelcco's
modified Eagle's minimal essential medium (DMEM) supplemented with
10% heat inactivated (56.degree. C., 30 minutes) fetal bovine serum
(FBS, Gibco BRL) and 1% antibiotics penicillin/streptomycin, in a
humidified atmosphere of 5% CO.sub.2 in air at 37.degree. C. P19
cells were cultured in minimal essential medium (MEM) supplemented
with the same FBS and antibiotic as above. Cells were sub-cultured
every two-three days (when confluent enough) by trypsinization.
[0031] DNA Transfection. Viruses were generated by co-transfecting
293T cells with five plasmids: pNHP, pTY, pHEF-VSV-G, pHEF-eGFP (as
transfection control), and a tat plasmid. A modified DNA
transfection protocol using the Superfect kit (Qiagen) was
performed. For a six well plate, cells were split exactly 17 hours
prior to transfection at about 90% confluency (9.times.10.sup.5 to
1.times.10.sup.6 cells per well). To be sure that the cells were
split without clumps, they were trypsin treated at 37.degree. C.
for 5 minutes. The next morning, the media was removed and the
cells were fed with 600 .mu.l of fresh growth DMEM with 10% FBS. In
an eppendorf tube were mixed: 75 .mu.l (per well) of serum free
DMEM; 2.7 .mu.g of helper DNA mix (1 .mu.g/.mu.l) containing 1.8
.mu.g of pNHP, 0.5 .mu.g of pHEF-VSV-G, 0.2 .mu.g of pCEP4tat, and
0.2 .mu.g of pHEF-eGFP; and 0.8 .mu.g of pTY DNA vector. After
vortexing, 7 .mu.l of Superfect (2:1 Superfect versus DNA) were
added to the center of the tube, and mixed immediately by pipeting
up and down five times. The mixture was then incubated at room
temperature for 5 to 10 minutes. To the six well culture plate
(with 600 .mu.l of growth media) the DNA mix was added dropwise.
The plate was then gently mixed by tilting back and forth a few
times, and incubated at 37.degree. C. in a humidified atmosphere of
3% CO.sub.2 in air for 4-5 hours. After incubation, the media was
removed, the cells were washed with the desired culture media and
fed with 1.5 ml of culture media per well. Virus was collected in
12 hours periods for three times (24 h, 36 h and 48 h) and stored
at -80.degree. C. for further use.
[0032] Virus Transduction and Titration. Virus supernatants were
filtered using a 0.45 .mu.m low protein-binding filter to remove
cell debris from transfected culture. The cells were split (TE671
or P19) at about 90% confluency and seeded in wells of a 24-well
culture plate. The cells were incubated at 37.degree. C. in a
humidified atmosphere of 5% CO.sub.2 for 2-4 hours or overnight.
The media was then removed from the cells, and 200-300 .mu.l/well
(just enough to cover the cells) of media containing 8 .mu.g of
polybrene/ml of media were added. (DMEM for TE671 and MEM for
P19).
[0033] For lentivirus titration, different volumes of virus stock
were used, usually, 1, 5 and 10 .mu.l for titer between 104 and 10.
These volumes of virus stock were added to the media and mixed by
swirling the plate. The cells were incubated at 37.degree. C. in a
humidified atmosphere of 5% CO.sub.2 overnight. The next day, 0.5
ml of growth media was added directly to the infected culture
without removing the old media. The cells were incubated in the
same conditions as above for 24 hours (the incubation can be up to
48 hours from the time the virus was added).
[0034] The cells were then assayed for nuclear lacZ enzyme. First,
the cells were washed twice with PBS and fixed for exactly 5
minutes at room temperature with 300 .mu.l of fixative solution
containing: 1% formaldehyde (0.27 ml of 37.6% for final 10 ml) and
0.2% glutaraldehyde (80 .mu.l of 25% for final 10 ml) in PBS. The
reaction was stopped by adding 500 .mu.l of PBS in each well. The
cells were then washed three times with PBS and incubated overnight
with the conditions described before in 300 .mu.l of staining
solution containing: 4 mM K-Ferrocyanide, 4 mM K-Ferricyanide, 2 mM
MgCl.sub.2 and 0.4 mg/ml X-Gal in PBS. The next day, the number of
blue cells were counted directly using an inverted microscope. The
best titer was usually observed for the virus harvested 36 hours
after transfection.
Example 2
Results
[0035] Lentiviral Vector Cosntruction. Lentiviral vectors carrying
an internal human elongation factor-1.alpha. (EF1 .alpha.)
promoter, a nuclear lacZ reporter gene, and cHS4 of SEQ ID NO:1
were constructed. The cHS4 fragment was inserted into the 3' LTR of
the lentiviral vector to generate three different constructs:
pTYcHS4-EFnLacZ forward, pTYcHS4-EFnLacZ reverse, and
pTYcHS4-EFnLacZ2xReverse (shown in FIG. 1C). During the reverse
transcription, the cHS4 element was copied into the 5' LTR of the
vector. Thus, as shown in FIG. 1C, when integrated into the host
chromosome, the reporter gene is flanked by the insulator.
[0036] As safety is a major concern with HIV-derived vectors, the
pNHP/pTY vector system was developed to minimize the possibility of
homologous recombination and replication competent virus (RCV)
production. To examine vector efficacy, human 293T cells were
co-transfected with the following five plasmids: pNHP; pHEF-VSV-G
(an envelope expression plasmid); pTYSalIEFnLacZ, pTYcPPTEFnLacZ
(controls), or one of the pTYcHS4 constructs shown in FIG. 1C (a
transducing pTY construct); pCEP4tat (a tat plasmid); and pHEFeGFP
(an internal transfection control). The vector titer was determined
by titration on TE671 and P19 cells using transfected culture
supernatants. The reporter gene LacZ was assayed by colorimetric
staining for .beta.-galactosidase activity. Results are shown in
Table 1.
[0037] As reported in Table 1, every construct successfully
produced viral vectors. The insulated vectors had titers close to
the control (pTYSalIEFnLacZ), demonstrating that the insulator
fragments had no adverse effect on the virus production. This was
not true for the construct with two copies of the cHS4 fragment,
probably because the fragment of about 500 bp affected the function
of the LTR and therefore the production of virus. The
pTYcPPTEFnLacZ is considered a control for this study because it
does not contain any insulator fragment. However, it contains a
cPPT sequence that has been cloned to increase the efficacy of the
vector transduction.
1TABLE 1 vector production in TE671, 36 h after transfection. (tu:
transducing unit) Vector TE671 titer (tu/ml) P19 titer (tu/ml)
pTYSaIIEFnLacZ 4.22 .times. 10.sup.6 2.87 .times. 10.sup.5
pTYcPPTEFnLacZ 1.23 .times. 10.sup.7 2.1 .times. 10.sup.6
pTYcHS4-EFnLacZ 3.16 .times. 10.sup.6 3.27 .times. 10.sup.5 forward
pTYcHS4-EFnLacZ 3.4 .times. 10.sup.6 2.64 .times. 10.sup.5 reverse
pTYcHS4EFnLacZ 6.72 .times. 10.sup.5 8 .times. 10.sup.4 2 .times.
reverse
[0038] Analysis of Long-Term Expression in Cells. To investigate
whether the cHS4 insulator can protect the transgene from the
silencing effect, a long-term in vitro study was carried out. Two
different cell types, TE671 and P19 cells were transduced with
either the controls without the insulator or one of the three
different constructs with the cHS4 element.
[0039] For each type of cell, two sets of transduction were carried
out. For one set, cells were transduced at 2 MOI (multiplicity of
infection), and for the other, they were transduced at 5 MOI in a
total of two rounds of infection. Transduced cells were grown until
confluent (4 days), trypsinized and plated into 6-well culture
plates. Later, they were cultured into T-25 flasks to avoid
contamination during handling. The transduced cells were
continuously propagated without selection. At different passage
times, some cells were frozen (for further experiments) and the
percentage of nLac Z expressing cells was determined.
[0040] Referring to FIGS. 2A and 2B, using TE671 cells, the
efficacy of transduction for all the constructs is about the same
at the beginning of the study, 4 days after the first infection. At
2 MOI, the efficacy is between 66% for the pTYcPPTEFnLacZ and 78%
for the pTYcHS4-EFnLacZ.
[0041] After 47 days of study, several things had been observed.
First, for the study at both 2 and 5 MOI, all the vectors displayed
decreased kinetics in the percentage of infected cells. The results
are consistent between the 2 MOI and the 5 MOI experiments. The
pTYcPPTEFnLacZ vector, which had the highest titer of infection
(see Table 1), appeared to infect the least number of cells at the
beginning of the study. The constructs with the highest decrease in
expression of the reporter gene were the two controls without cHS4
modification. This decrease of expression is about 33% at 2 MOI and
28% at 5 MOI. This suggests that the silencing effect occurred in
these cells.
[0042] The decrease of expression of the transgene 47 days after
infection is shown in FIG. 3. The construct with two copies of the
cHS4 insulator appears to be the one that protects the expression
of the reporter gene the best. The decrease of expression was only
about 14.5% at 2 MOI and 15.6% at 5 MOI, which is half of what was
observed for the two control constructs. It was also observed that
the construct with a single copy of the cHS4 insulator in forward
orientation seems to work better than the construct with a single
copy of the cHS4 insulator in reverse orientation.
[0043] The same long-term study was carried out on P19 mouse
embryonic stem cells. In previous studies, embryonic cells had been
shown to have a silencing effect only three days after infection.
This is probably due to their strong regulation system that allows
them not to differentiate. Referring to FIGS. 4A and 4B, only four
days after the first infection, the difference in the expression of
insulated transgene and the control was already significant. A
further experiment was performed to confirm these results. At day
0, the P19 cells were transduced at 5 MOI into a twelve well
culture plate. At day 1, 24 hours after the first infection, a part
of the cells was sampled for Lac Z assay and the percentage of cell
transduced was determined. The result of this short-term study
showed that all the constructs, including the control, transduced
the P19 cells with the same efficiency.
[0044] In an additional experiment, another portion of the cells
was transduced a second time at 5 MOI and cultured. A second Lac Z
assay was then performed 56 hours after the first infection. At
this time point, the cells were transferred in a six well plate and
LacZ assayed each time they were confluent (every three days). As
shown in FIG. 5, after the second infection, a large number of
cells were transduced and a difference between the insulated
constructs and the control was observed. This demonstrated that the
difference of transgene expression observed four days after
infection was attributable to the activity of the insulator, rather
than because more cells were transduced at the beginning. This also
showed that the P19 cells were not transduced with the same
efficacy as the TE671, even with two rounds of infection at 5 MOI.
After the second infection, a higher percentage of infected cells
was obtained for the pTYcHS4EFnLacZ forward with only 47% (when the
lowest percentage observed in TE671 was at 2 MOI for the
pTYcPPTEFnLacZ with 67% of transduced cells). The construct with
two copies of the cHS4 in reverse orientation does not seem to
insulate silencing (contrary to what was observed in the TE671
cells).
[0045] The gradual loss of transgene expression observed might have
been due to transgene silencing or the loss of transduced cells. To
distinguish between these two mechanisms, early and late passages
of the same transduced cells were compared by Southern blot
analysis using a control (pTYSalI) and an insulator vector
(pTYcHS4Forward) to transduce both TE671 and P19. The genomic DNA
was harvested and quantified and the same amount of DNA for each
sample was used in the analysis. The results showed that within
fifteen passages of TE671 cells, there was little to no loss of the
integrated lentiviral transgene for both wild type and insulator
vector transduced cells. However, P19 cells clearly demonstrated a
rapid loss of lentiviral transgene after fifteen passages for cells
transduced with either the wild type vector (pTYSalI) or the
insulator vector (pTYcHS4Forward).
Other Embodiments
[0046] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
1 1 240 DNA Unknown Store Bought Purdue Chicken 1 gagctcacgg
ggacagcccc ccgccaaagc ccccagggat gtaattgcat ccctcttccg 60
ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgcttc ccccgcatcc
120 ccgcgagccg agccggcagc gtgcggggac agcccggcac ggggaaggtg
gcacgcgatc 180 gtttcctctg aacgcttctc gctgctcttt gagcctgcag
acacctgggg ggatacgggg 240
* * * * *